Novel therapeutic target and diagnostic marker for asthma and related conditions

ABSTRACT

CD48, a surface-marker molecule present in eosinophils, is disclosed herein as a key molecule in allergic conditions, particularly in allergic airway inflammations like asthma, allergy and nasal polyposis. CD48 is thus presented as a target molecule in the treatment of said conditions. In addition, diagnostic methods, and a kit for the diagnosis of allergic inflammatory conditions are described, based on the detection of CD48 expression.

FIELD OF THE INVENTION

The present invention relates generally to the field of allergicconditions. In particular, the present invention is concerned withproviding a marker for the diagnosis of allergic inflammation,particularly, allergic airway inflammation such as asthma and relatedconditions, as well as with agents for the treatment of said conditions.

BACKGROUND OF THE INVENTION

All publications mentioned throughout this application are fullyincorporated herein by reference, including all references citedtherein.

Asthma is a chronic and complex inflammatory disease of the airwayscharacterized by airflow obstruction, bronchial hyper-responsiveness(BHR) and airway inflammation. It is the most common chronic illness ofchildhood, with up to 20% of children affected in some Westerncountries. The incidence as well as the number of hospital admissionsattributable to asthma continues to rise in both adults and children.Over the last decade the importance of airway inflammation in thedisease process has been carefully investigated, and revealed that theasthmatic tissue is characterized by the accumulation of a large numberof inflammatory cells (e.g. eosinophils, neutrophils, basophils, mastcells), increased mucus production, epithelial shedding and hypertrophy,mucus, smooth muscle cell hypertrophy and sub-mucosal mucus glandshyperplasia/metaplasia and fibrosis. Notably, chronic inflammation ofthe asthmatic lung leads to structural changes, that in turn exacerbatethe hyperresponsiveness observed in this disease. While these findingshave provided the rationale for the development of multiple therapeuticagents that interfere with specific inflammatory pathways, thedevelopment of the asthma phenotype is likely to be related to a complexinterplay of a large number of genes combined with environmentalfactors.

Recent genome searches have revealed that at least 19 genes contributeto asthma susceptibility and microarray studies of asthmatic tissuerevealed the involvement of hundreds of genes. Moreover, microarrayanalysis has recently demonstrated increased expression of 291 genesthat were commonly involved in murine disease pathogenesis rather thanto a particular mode of disease induction [Zimmermann, N. et al. (2003)J. Clin. Invest. 111: 1863-74]. Therefore, a central issue still underpursuit is identification of fundamental molecules/pathways that governthe processes underlying inflammation in asthma. Nonetheless, no asthmadrug so far has been able to inhibit the initial steps of the signalingcascade of this agonizing medical condition. Therapeutic drugs forasthma are usually directed to the symptoms, i.e., ex post facto of theasthma attack.

Eosinophils are thought to be key effector cells in asthma by therelease of basic granule proteins, membrane phospholipid metabolites anda variety of cytokines. For example, the eosinophil basic proteins havebeen found to be highly toxic in vitro to respiratory epithelial cells,at concentrations detected in biological fluid from patients withasthma. Furthermore, eosinophils produce matrix metalloproteinase(MMP)-9, tissue inhibitor of matrix metalloproteinase (TIMP)-1/2,contain heparanase and are a source for vascular endothelial growthfactor (VEGF), platelet derived growth factor (PDGF) and b-fibroblastgrowth factor (b-FGF), indicating their role in asthma-associatedsymptoms.

Since a central question concerning eosinophils is to understand themechanisms by which these cells get activated or inhibited and whether a“disease-specific” activator exists, the inventors screened eosinophilswith monoclonal antibodies (mAbs) produced to recognize epitopesexpressed on T lymphocytes and NK cells, preferably. The inventors foundthat human eosinophils express 2B4 [Munitz, A. et al. (2005) J. Immunol.174: 110-118], and that 2B4 is a functional activator receptor on thesecells in vitro, suggesting that a complex network of activating signalsregulate the immunological or inflammatory responses coordinated byeosinophils.

CD48 is a glycosyl-phosphatidyl-inositol (GPI) anchored proteinbelonging to the CD2-subfamily, which is involved in lymphocyteadhesion, activation and co-stimulation. It is expressed mainly onhematopoietic cells and exists in both a membrane-associated and asoluble form. Studies on CD48-deficient mice indicate that CD48 has abroad immunological importance. In fact, CD48 has been described tointeract with extracellular matrix components such as heparin sulfate,facilitate cell adhesion, innate responses to bacterial infection andgraft rejection and provide co-stimulatory signals to T and Blymphocytes. Furthermore, CD48 has a distinctive role in orchestratingmast cell innate responses towards E. coli. Besides, CD48 is a lowaffinity ligand for CD2 but a high affinity ligand for 2B4 [Brown, M. H.et al. (1998) J. Exp. Med. 188(11):2083-90]. CD48-2B4 interactions canmodulate T cell, B cell and NK cell functions and cross-talk. Studieswith 2B4 gene-targeted mice, demonstrated that 2B4-CD48 interactions areessential for expansion and activation of murine NK cells. The absenceof functional 2B4-CD48 interactions impairs NK cell cytotoxic responseand IFN-γ release upon tumor target exposure. Furthermore, activated NKcells significantly increase the CD3-dependent proliferation of CD8⁺ andCD4⁺ T cells by a 2B4-CD48 dependent mechanism. n. Furthermore,cross-linking of CD48 on the surface of rodent T lymphocytes inducedmobilization of the intracellular calcium inositol triphosphateconcentration. T cell activation via CD48 combined with CD3 inducedenhanced IL-2 release, T cell receptor signaling and cytoskeletalreorganization. Furthermore, cross-linking of CD48 on the surface of rator murine B cells induced strong homotypic adhesion suggesting that thismolecule can be involved in B cell activation. In humans, cross-linkingof CD48 on purified tonsillar B cells significantly increasedCD40-mediated activation. Additionally, CD48, in combination with IL-4and/or IL-10 is able to induce B cell aggregation, proliferation and IgGsecretion [Klyushnenkova, E. N. et al. (1996) Cell Immunol.174(1):90-8].

SUMMARY OF THE INVENTION

In view of the fact that allergic airway condition and specifically,asthma is a Th2 associated process, it is worthwhile mentioning thatuntil the present invention, the contribution of CD48 has not beenexplored in Th2 settings. In this study, the inventors investigated thecontribution of CD48 and its ligands to allergic eosinophilic airwayinflammation. Therefore, the present invention show for the first timethat CD48 is up regulated in two murine models of allergic eosinophilicairway inflammation. More particularly, the present inventiondemonstrates that CD48 is up-regulated in two mouse models ofexperimental asthma. The findings indicate that CD48 over-expression isat least partially regulated by IL-3. Furthermore, neutralization ofCD48 in allergen-challenged mice resulted in abrogation of lunginflammation, airway smooth muscle thickening, mucus production andgoblet cell hyperplasia as well as in decreased eosinophilia in the BALF(bronchoalveolar lavage fluid) and spleen. In addition, neutralizationof CD48 diminished chemokine and cytokine levels in the BALF. Finally,the expression of CD48 was up-regulated in eosinophils from atopicasthmatics vs. normal controls, pointing out the critical involvement ofthis molecule in asthma. Furthermore, experiments with anti-CD48,anti-CD2 and anti-2B4 neutralizing mAbs, demonstrate that CD48 iscritically involved in allergic eosinophilic airway inflammation.

Thus, the present invention provides CD48 as a molecular marker and as atarget for therapeutic and diagnostic methods for allergic conditions,specifically, asthma and asthmatic related conditions.

It is therefore one object of the invention to provide methods oftreating an allergic condition, specifically, an allergic-eosinophilicairway inflammation such as asthma and asthma related disorders, byspecifically inhibiting the activity or the expression of CD48.

Another object of the invention relates to a therapeutic composition forthe treatment of allergic inflammation, particularly, allergic airwayinflammation, using an anti-CD48 agent such as an anti-CD48 antibody, anantibody against a down-stream molecule of the CD48 stimulatory pathway,or a nucleic acid molecule specifically targeted to decrease CD48expression or to decrease the expression of a down-stream membermolecule of the CD48 stimulatory pathway.

In another object, the invention provides a diagnostic method fordetection of an allergic condition in a subject. This method involvesdetermining CD48 levels of expression by a suitable means either in theprotein or nucleic acids level. Whereby elevated expression of CD48 incomparison to a negative control, indicates the presence of suchcondition.

Other objects, purposes and advantages of the invention will becomeapparent as the description proceeds.

In a first aspect, the invention relates to a method of treating anallergic condition, particularly, an allergic inflammation,specifically, airway inflammation such as asthma and nasal polyposis,atopic dermatitis, conjunctivitis and intestinal allergy. This methodcomprises administering a therapeutically effective amount of ananti-CD48 agent which blocks CD48 stimulatory pathway, to a subject inneed of such treatment.

According to one embodiment, the anti-CD48 agent used by the method ofthe invention preferably blocks CD48 stimulatory pathway by inhibitingthe activity and/or the expression of CD48 or of a down-stream membermolecule of the CD48 stimulatory pathway. The anti-CD48 agent used bythe invention may be a protein-based molecule, such as anti-CD48antibody, which specifically binds to CD48.

The anti CD48 agent may also be an antagonist of the CD48 stimulatorypathway, capable of decreasing or blocking an agonist of said pathway,in a competitive or non-competitive manner.

Alternatively, the anti-CD48 agent may be a nucleic acid based moleculesuch as, antisense oligonucleotide specific to the CD48 sequence, aribozyme having catalytic activity (such as cleavage) that renders theCD48 inactive, an interfering RNA (RNAi) such as small interfering RNA(siRNA), or a microRNA capable of preventing the expression(transcription and translation, respectively) of the CD48 into protein.

The invention further provides a method of inhibiting the activityand/or expression of CD48 in cells of a subject in need thereof. Thismethod comprises the step of in vivo contacting the cells with aneffective amount of an anti-CD48 agent.

According to another aspect, the invention relates to a method for thediagnosis of an allergic condition in a subject. The diagnostic methodof the invention comprises the steps of: (a) obtaining a biologicalsample from said subject; and (b) determining the level of expression ofCD48 in said biological sample by a protein-based or a nucleicacid-based detection method. It should be appreciated that elevatedexpression of CD48, in comparison with negative control, indicates thepresence of an allergic condition.

According to one embodiment, the CD48 expression may be detected by aprotein-based method comprising the steps of: (i) contacting theexamined sample with a CD48 binding agent, preferably, an anti-CD48antibody; and (ii) measuring the level of binding of the agent to theCD48 protein in the sample by a suitable protein based detection assay.As a non-limiting example, such assay may be immunohistochemicalstaining, Western blot analysis, immunopercipitation, flow cytometry,ELISA, competition assay, any combination thereof or any other suitableassay.

According to another embodiment, the CD48 expression may be detected bya nucleic acid based detection method such as in-situ hybridization,RT-PCR, nucleic acid based ELISA, RNAse protection assay, Northern blotanalysis, any combination thereof or any other nucleic-acid basedsuitable assay.

In yet another aspect, the invention relates to a kit for the diagnosisof an allergic condition, the kit comprising as follows: an agent fordetermining the presence of an analyte of interest, wherein said analyteof interest is one of CD48 protein or CD48 mRNA. The kit furthercomprises calibration means.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-1C: DNA microarray analysis identifies CD48 as anallergen-induced gene in allergic eosinophilic airway inflammation

Expression of CD48 in Ovalbumin (OVA) (A) and A. fumigatus-challengedmice (B) as measured by gene chip analysis is shown. The averagedifference for the hybridization signal after saline (gray bar) andallergen (black bar) challenge is depicted (n=3 for Aspergillus controlgroup and n=4 for OVA control group and OVA and Aspergillus experimentalgroups). The induction of CD48 in allergen-challenged mice as measuredby Northern blot analysis is shown (C). Total RNA was electrophoresed,transferred and hybridized with a radiolabeled sequence-confirmed CD48cDNA probe. The location of 18S RNA is shown. Each lane represents anextract from one separate mouse.

FIG. 2A-2B: Peripheral blood eosinophils and nasal polyp eosinophils ofatopic asthmatics display enhanced levels of CD48 expression

Human peripheral blood eosinophils (A) and human nasal polyp eosinophils(B) from atopic asthmatic (n=7 and 8, respectively) or normal (n=6 and9, respectively) individuals were stained with anti-CD48 mAb followed bygoat anti-mouse FITC and analyzed by FACS. For identification of nasalpolyp eosinophils, the cells were additionally stained with ratanti-mouse CCR3 PE. CCR3⁺/SSC^(high) cells were identified aseosinophils and analyzed for CD48 expression. Data are presented as meanfluorescent intensity (MFI), each dot represents one donor.

*, p<0.05; ** p<0.005.

FIG. 3A-B: The expression of CD48 on human peripheral blood eosinophilsis regulated by IL-3

Human peripheral blood eosinophils were incubated with the indicatedconcentrations of rhIL-3, rhIL-5 or rhGM-CSF (A) for 18 hours.Thereafter, cells were washed twice, stained with anti-CD48 mAb followedby goat anti-mouse FITC and analyzed by FACS. Kinetic analysis (B) wasperformed by incubating human peripheral blood eosinophils with 20 ng/mlof rhIL-3 for the indicated time points. Thereafter, cells were assessedfor CD48 expression as described above. Data are presented as meanfluorescent intensity (MFI), each dot represents one donor.

*, p<0.05; ** p<0.005, n=5.

FIG. 4A-E: CD48 expression is independent of STAT6, IL-4 and IL-13

RNA was extracted from the lungs of wild-type (A-D, left panel), STAT6deficient [A-B, right panel], IL-13 and IL-4/IL-13 deficient mice (C-D,middle and right panels respectively). Mice that express atetracycline-inducible IL-13 were fed doxycycline-containing food forthe indicated time periods (E). For all Northern blot assays, total RNAwas electrophoresed, transferred and hybridized with a radiolabeledsequence confirmed CD48 cDNA probe. The location of 18S RNA is shown.Each lane represents an extract from one separate mouse, EtBr=ethidiumbromide.

FIG. 5: CD48 activates human peripheral blood eosinophils to release EPO(eosinophil peroxidase)

Human peripheral blood eosinophils were cultured in wells pre-coatedwith sheep anti-mouse F(ab)₂ Ab and either isotype-matched control(isotype) or anti-CD48 mAb (anti-CD48) for 30 min. Culture supernatantswere collected and EPO concentration was determined by a colorimetricassay. The data represent the mean±SD of three different experimentsperformed in triplicate.

***, p<0.001, n=3.

FIG. 6A-G: IL-3 regulates CD48 expression in vivo IL-3 mixed withanti-IL-3 mAb at a 2:1 molar ratio, [IL-3C] was administered intranasal(A-D) or systemically (E-F) every other day for 21 days to normal BALB/cmice. Twenty-four hours after the last IL-3C administration, mice weresacrificed, BALF was performed and lungs and spleen excised fordifferential cell counts as assessed by FACS analysis (A-B, E). Forassessment of CD48 expression (C-D, F), BALF and lung cells were stainedadditionally with anti-CD48 mAb and evaluated by FACS. Eosinophils fromthe spleen of IL-5 transgenic or wild type mice (G) were stained withanti-CD48 mAb and evaluated by FACS. For (A-B,E) data are presented astotal cell number±SD; for (C-D and E-F) data are presented as meanfluorescence intensity [MFI]±SD, from 4-6 mice per group.

*, p<0.05; ** p<0.005, ***, p<0.0001, n=3.

FIG. 7A-B: CD48 is upregulated on murine eosinophils in experimentalasthma and in allergic peritonitis

OVA/alum sensitized mice were challenged with OVA. Mice were sacrificedat the indicated time points after the last allergen challenge and BALF(A) or peritoneal lavage (B) was performed. The cells were stained withPE-labeled anti-CD48 and FITC-labeled anti-CCR3. CCR3⁺/SSC^(high) cellswere gated and analyzed for CD48 expression. Data are expressed as meanfluorescence intensity [MFI]±SD from 4-6 mice per group.

*, p<0.05; ** p<0.005, n=3.

FIG. 8A-C: Neutralization of IL-3 in murine experimental asthma reducesCD48 expression

OVA/alum sensitized mice were treated with anti-IL-3 mAb or anisotype-matched control mAb (2 mg/mouse) on day 23 (24 hrs beforeallergen challenge) and days 24 and 27 (1 hr before allergen challenge).BALF was performed and lungs were excised, 24 hrs after the lastallergen challenge. BALF cells were stained with PE-labeled anti-CD48and FITC-labeled anti-CCR3. CCR3⁺/SSC^(high) cells were gated andanalyzed for CD48 expression (A) and percentage of eosinophils, definedas CCR3⁺/SSC^(high) cells (B). Lungs were fixed, paraffin embedded,stained for H&E and scored as described (C). In (A and B) data areexpressed as mean fluorescence intensity [MFI]±SD or percentage ofcells, respectively. In (C) data are expressed as mean inflammatoryscore±SD. All data were obtained from 4-6 mice per group.

n=3*, p<0.05.

FIG. 9: Cellular source of CD48, CD2 and 2B4 in the lungs

OVA/alum sensitized mice were challenged with OVA. Eighteen hours afterthe last allergen challenge the lungs were harvested and expression ofCD48, CD2 and 2B4 was analyzed on various cell types. The data arerepresented as ΔMFI±SD n=3. Eos—eosinophils, Neut—neutrophils,Lymph—CD4⁺ lymphocytes, Mac—macrophages, NK—NK cells, NKT—NKT cells,parentheses indicate the percentage of the indicated cell in the BALF.

FIG. 10A-G: Neutralization of CD48 attenuates eosinophilic inflammation,Th2 and proinflammatory cytokines expression in the BALF

OVA/alum sensitized mice were treated with anti-CD48, anti-CD2 oranti-2B4 mAbs or control antibodies (Rat IgG, Hamster IgG) on day 23 anddays 24 and 27 one hour before allergen challenge (250 μg per mice).Twenty four hours after the last allergen challenge BALF was performedand the cells were stained for differential cell identification.CCR3⁺/VLA4⁺/CD3⁻/SSC^(high) cells were gated and considered aseosinophils (A). Assessment of IL-4, IL-5, IL-13, TNF-α, eotaxin-1 (B-Frespectively) in the BALF was detected by ELISA according to themanufacturers' instructions. Data are represented as mean±SD of n=3 (4-6mice/group/n). Anti-CD48, anti-CD2 or anti-CD48 antibodies wereadministered systemically to naïve Balb/c mice (250 μg/mouse). After 24hrs the mice were sacrificed and differential cell population in thespleen and peripheral blood were monitored. A histogram plot analysis ofspleen cell populations is shown (G). Data are expressed as mean±SD,n=3.

FIG. 11A-D: Neutralization of CD48 attenuates lung inflammation

Mice were sensitized, challenged and treated as described. The lungtissue was fixed, paraffin embedded and stained with H&E for assessmentof inflammation. Representative photomicrographs (magnification: ×40) ofairway inflammation in the different treatment groups (A). Quantitativeanalysis of alveolar space (B), peribronchial inflammation (C) and lungperivascular (D) is presented. Data are the mean±SD of n=3 (4-6mice/group/n).

FIG. 12A-C: Neutralization of CD48 attenuates goblet cell hyperplasia,mucus production and smooth muscle thickening in the lung.

Mice were sensitized, challenged and treated as described. The lungtissue was fixed, paraffin-embedded and stained with PAS for assessmentof goblet cell hyperplasia and mucus production. A representativephotomicrograph (magnification: ×40) of airway PAS staining in thedifferent mice treatments (A). Quantitative analysis of PAS⁺ cells inthe bronchial epithelium (B). Peribronchial smooth muscle thickening wasanalyzed (C). Open circles represent the mean peribronchial smoothmuscle thickness in pixels of three mid sized bronchioles per mouse.Data are the mean±SD of n=3 (4-6 mice/group/n).

DETAILED DESCRIPTION OF THE INVENTION

The present study is the first demonstration (both in humans and mice)that CD48 is expressed in eosinophils and its expression is directlycorrelated with the triggering of the asthmatic response, as detailedbelow. Furthermore, it is also the first report implicating CD48 inallergy, and the allergic response.

Thus, in a first aspect, the present invention provides a method oftreating an allergic condition, specifically, allergic airwayinflammation such as asthma or nasal polyposis, atopic dermatitis,conjunctivitis and intestinal allergy. The method of the inventioncomprises administering a therapeutically effective amount of ananti-CD48 agent that blocks CD48 stimulatory pathway, to a subject inneed of said treatment.

In one specific embodiment, said anti-CD48 agent is an anti-CD48antibody.

In a more particular aspect, the present invention provides a method ofinhibiting the activity and/or expression of CD48 in cells of a subjectsuffering from an allergic condition, wherein said method comprises invivo contacting said cells with an effective amount of an anti-CD48agent.

It should be appreciated that the invention further encompasses ex vivocontacting said cells with the anti-CD48 agent.

Thus, as mentioned above, the present invention relates to a method forthe treatment of allergic conditions such as asthma, atopic dermatitis,conjunctivitis, intestinal allergy, and nasal polyposis, throughabrogating, inhibiting or decreasing CD48 expression, or througheliminating, reducing or neutralizing CD48 activity. Such treatmentcomprises administering a therapeutically effective amount of ananti-CD48 agent to a subject in need.

As used herein, the term “anti-CD48 agent” or “an agent that blocks CD48stimulatory pathway” refers to any compound, complex or compositionwhich reduces, or even abrogates, the physiological phenomena which arecaused by, or are downstream from CD48 activation. More particularly,such agent reduces or even inhibits the stimulatory signaling by othermember molecules which are down-stream from the CD48 molecule, in suchpathway which is involved in the allergic inflammatory condition. Saidagent may be effective at various levels: firstly at the mRNA expressionlevel, where CD48 (or any other down-stream member molecule) expressionmay be reduced (or inhibited) by inhibiting its transcripts e.g. throughspecific antisense oligonucleotides or by various modes of interferingRNAs, such as RNAi, siRNA, microRNA and ribozyme, which more directlyinterfere with translation.

Antisense oligonucleotides refer to a nucleotide comprising essentiallya reverse complementary sequence to a sequence of CD48 mRNA. Thenucleotide is preferably an oligodeoxynucleotide, but alsoribonucleotides or nucleotide analogues, or mixtures thereof, arecontemplated by the invention. The antisense oligonucleotide may bemodified in order to enhance the nuclease resistance thereof, to improveits membrane crossing capability, or both. The antisense oligonucleotidemay be linear, or may comprise a secondary structure. It may alsocomprise enzymatic activity, such as ribozyme activity.

By “ribozyme” it is meant an RNA molecule which has complementarity in atarget binding region to a specified gene target, for example CD48, andalso has an enzymatic activity which is active to specifically cleavetarget RNA. Said molecule is capable of catalyzing a series of reactionsincluding the hydrolysis of phosphodiester bonds in trans (and thus cancleave other RNA molecules) under physiological conditions. Suchenzymatic nucleic acid molecules can be targeted to virtually any RNAtranscript, and achieve efficient cleavage in vitro. That is, theenzymatic RNA molecule is able to intermolecularly cleave RNA andthereby inactivate a target RNA molecule. The complementary regionsallow sufficient hybridization of the enzymatic RNA molecule to thetarget RNA and which ensures specific cleavage. One hundred percentcomplementarity is preferred, but complementarity as low as 50-75% mayalso be useful in this invention. The nucleic acids may be modified atthe base, sugar, and/or phosphate groups.

The term “siRNAs” refers to short interfering RNAs. The term “RNAinterference” or “RNAi” refers to the silencing or decreasing of geneexpression by siRNAs. It is the process of sequence-specific,post-transcriptional sequence-specific gene silencing in animals andplants, initiated by siRNA that is homologous in its duplex region tothe sequence of the gene to be silenced, particularly CD48. Theexpression of the gene is either completely or partially inhibited. RNAimay also inhibit the function of a CD48 RNA, and said function may becompletely or partially inhibited.

By “microRNA” is meant, single-stranded RNA molecules of about 21-23nucleotides in length thought to regulate the expression of other genes.miRNAs are encoded by genes that are transcribed from DNA but nottranslated into protein (non-coding RNA), instead they are processedfrom primary transcripts known as pri-miRNA to short stem-loopstructures called pre-miRNA and finally to functional miRNA. MaturemiRNA molecules are complementary to regions in one or more messengerRNA (mRNA) molecules, which they target for degradation.

Alternatively, CD48 activity may be reduced through the use of anantagonist of the receptor, a partial antagonist or an antibody thateither competes with the natural agonist, blocks the activity orencourages the uptake of the CD48 molecule. The anti-CD48 agent may thusbe a nucleic acid sequence, a protein, a peptide, an antibody or a smallorganic molecule.

Thus, as mentioned above, a preferred anti-CD48 agent is an anti-CD48antibody, but other agents may be effective as well, such asCD48-specific siRNA, RNAi, microRNA and Ribozyme. Other CD48 inhibitorsand/or antagonists include anti-CD48 specific antibody fragments(F(ab′)2 or Fab′), single chain Fv, and Fc-fusion protein of CD48ligands, e.g. Fc fusion proteins of 2B4 or CD2.

The anti-CD48 antibody used in the method of treatment may be ofpolyclonal or monoclonal origin. A monoclonal antibody may be improved,through a humanization process, to overcome incompatibility problems.Rapid new strategies have been developed recently for antibodyhumanization which may be applied for such antibody. These technologiesmaintain the affinity, and retain the antigen and epitope specificity ofthe original antibody [Rader, C., et al. (1998) Proc. Natl. Acad. Sci.USA. 95: 8910-8915; Mateo, C. et al. (1997) Immunothechnology 3: 71-81].A “humanized” antibody, in which, for example animal (say murine)variable regions are fused to human constant regions, or in which murinecomplementarity-determining regions are grafted onto a human antibody.Unlike, for example, animal-derived antibodies, “humanized” antibodiesoften do not undergo an undesirable reaction with the immune system ofthe subject.

Thus, as used herein, the term “humanized” and its derivatives refers toan antibody which includes any percent above zero and up to 100% ofhuman antibody material, in an amount and composition sufficient torender such an antibody less likely to be immunogenic when administeredto a human being. It is being understood that the term “humanized” readsalso on human derived antibodies or on antibodies derived from non-humancells genetically engineered to include functional parts of the humanimmune system coding genes, which therefore produce antibodies which arefully human.

As referred to herein, CD48 expression may be understood as the presenceof CD48 protein or mRNA in the cells of interest, particularlyleucocytes, more particularly eosinophils. It should be appreciated thatthe invention further encompasses the presence of CD48 in any body fluidsample and in any immune-system cell (particularly the Th2 associatedcells).

The expression of CD48 was evaluated on human peripheral bloodeosinophil after incubation with various cytokines and chemokines, amongthem IL-3, IL-5 and GM-CSF, which are eosinophil survival cytokines andchief regulators of eosinophil functions, including priming andactivation [Giembycz, M. A. and Lindsay, M. A. (1999) Pharmacol. Rev.51: 213-340]. These cytokines share a βC-chain that is responsible foractivating their signaling pathways and consequent effects.

As per the results presented herein, elevated levels of CD48 expression,as compared to a negative control, provide a positive diagnosis of anallergic condition such as asthma, allergy (atopic dermatitis,conjunctivitis and intestinal allergy), or nasal polyposis, whereas low(or absent) levels of CD48 expression may be interpreted as a negativediagnosis of the same.

Thus, a further aspect of the present invention concerns a method forthe diagnosis of an allergic condition and particularly of asthma andrelated conditions, said method comprising obtaining a biological samplefrom the examined subject and determining the level of expression ofCD48 in the sample. Elevated expression of CD48, in comparison withnegative control, indicates the presence of asthma or an asthma-relatedcondition. Preferably, the biological sample may be a body fluid samplesuch as leucocyte-containing body fluid, blood, lymph, milk, urine,faeces, semen, appendix, spleen, extractstears, sputum, nasal, mucus,amniotic fluid, bronchoalveolar lavage, pleuric fluid, peritoneal fluidand tonsillar tissue extractstears, more preferably, aleucocyte-containing body fluid. It should be noted that theleucocyte-containing body fluid may be any body fluid, for example,blood, lymph, milk, urine, faeces, semen, appendix, spleen,extractstears, sputum, nasal, mucus, amniotic fluid, bronchoalveolarlavage, pleuric fluid, peritoneal fluid and tonsillar tissueextractstears.

Said diagnostic method of the invention may be applied at two levels. Inone embodiment, CD48 is detected at the protein level, in which casesaid method comprises contacting a biological sample with a CD48 bindingagent and measuring the level of binding of said agent to said CD48protein, whereby elevated binding of CD48, in comparison with a negativecontrol, indicates the presence of said allergic condition. Preferably,said CD48 binding agent is an anti-CD48 antibody.

Detection of CD48 at the protein level may be effected through variousmeans, including the detection of CD48 in intact cells, as in e.g. FACSanalysis, ELISA Spot Assay or immunohistochemistry, or through anymethodology which involves cell lysis and protein detection, such ase.g. Western Blot, ELISA, RIA, etc. All these methodologies are wellknown to the man skilled in the art, and have been described, e.g., inCurrent Protocols in Immunology, Coligan et al. (eds), John Wiley &Sons. Inc., New York, N.Y., 1999.

It should be noted that the term “antibody” as used herein throughoutthis specification is meant to include intact molecules as well asfragments thereof, such as, for example, Fab and F(ab′)₂, which arecapable of binding antigen, in this case the CD48 molecule (or protein).Such antibody fragments are within the scope of the present inventionand may be used for the kits and the treatment and diagnostic methodsdisclosed herein for intact antibody molecules, to the extent where saidfragments have the same biological activity, e.g. inhibition of CD48function/expression, or recognition of the CD48 protein, as the intactantibody. Such fragments are typically produced by proteolytic cleavage,using enzymes such as papain (to produce Fab fragments) or pepsin (toproduce F(ab′)₂ fragments).

Said antibody may be of polyclonal or monoclonal antibody. Methods ofpreparing polyclonal and monoclonal antibodies are well known to the manskilled in the art.

The biological sample used by the invention may be treated with a solidphase support or carrier such as nitrocellulose, or other solid supportor carrier which is capable of immobilizing cells, cell particles orsoluble proteins. The support or carrier may then be washed withsuitable buffers followed by treatment with a detectably labeledantibody in accordance with the present invention, as noted above. Thesolid phase support or carrier may then be washed with the buffer asecond time to remove unbound antibody. The amount of bound label onsaid solid support or carrier may then be detected by conventionalmeans.

By “solid phase support”, “solid phase carrier”, “solid support”, “solidcarrier”, “support” or “carrier” is intended any support or carriercapable of binding antigen (CD48) or antibodies against CD48. Well-knownsupports or carriers, include glass, polystyrene, polypropylene,polyethylene, dextran, nylon amylases, natural and modified celluloses,polyacrylamides, and magnetite. The nature of the carrier can be eithersoluble to some extent or insoluble for the purposes of the presentinvention. The support material may have virtually any possiblestructural configuration so long as the coupled molecule is capable ofbinding to an antigen or antibody. Thus, the support or carrierconfiguration may be spherical, as in a bead, cylindrical, as in theinside surface of a test tube, or the external surface of a rod.Alternatively, the surface may be flat such as a sheet, test strip, etc.Preferred supports or carriers include polystyrene beads. Those skilledin the art will know many other suitable carriers for binding antibodyspecific for CD48 or the CD48 antigen, or will be able to ascertain thesame by use of routine experimentation.

The binding activity of a given lot of antibody, of the invention asnoted above, may be determined according to well known methods. Thoseskilled in the art will be able to determine operative and optimal assayconditions for each determination by employing routine experimentation.

Other such steps as washing, stirring, shaking, filtering and the likemay be added to the assays as is customary or necessary for theparticular situation.

One of the ways in which an anti-CD48 antibody in accordance with thepresent invention can be detectably labeled is by linking the same to anenzyme and used in an enzyme immunoassay (EIA). This enzyme, in turn,when later exposed to an appropriate substrate, will react with thesubstrate in such a manner as to produce a chemical moiety which can bedetected, for example, by spectrophotometric, fluorometric or by visualmeans. Enzymes which can be used to detectably label the antibodyinclude, but are not limited to, malate dehydrogenase, staphylococcalnuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. The detection can be accomplished by colorimetricmethods which employ a chromogenic substrate for the enzyme. Detectionmay also be accomplished by visual comparison of the extent of enzymaticreaction of a substrate in comparison with similarly prepared standards.

Detection may be accomplished using any of a variety of otherimmunoassays. For example, by radioactive labeling the antibodies orantibody fragments, it is possible to detect receptor tyrosinephosphatase (R-PTPase) through the use of a radioimmunoassay (RIA). Agood description of RIA may be found in Laboratory Techniques andBiochemistry in Molecular Biology, by Work, T. S. et al., North HollandPublishing Company, NY (1978) with particular reference to the chapterentitled “An Introduction to Radioimmune Assay and Related Techniques”by Chard, T., incorporated by reference herein. The radioactive isotopecan be detected by such means as the use of a γ counter or ascintillation counter or by autoradiography.

It is also possible to label an antibody in accordance with the presentinvention with a fluorescent compound. When the fluorescently labeledantibody is exposed to light of the proper wavelength, its presence canbe then detected due to fluorescence. Among the most commonly usedfluorescent labeling compounds are fluorescein isothiocyanate,rhodamine, phycoerythrine, pycocyanin, allophycocyanin, o-phthaldehydeand fluorescamine.

The antibody can also be detectably labeled using fluorescence emittingmetals such as ¹⁵²E, or others of the lanthanide series. These metalscan be attached to the antibody using such metal chelating groups asdiethylenetriamine pentaacetic acid (ETPA).

The antibody can also be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

Likewise, a bioluminescent compound may be used to label the anti-CD48antibody used by the diagnostic method of the invention. Bioluminescenceis a type of chemiluminescence found in biological systems in which acatalytic protein increases the efficiency of the chemiluminescentreaction. The presence of a bioluminescent protein is determined bydetecting the presence of luminescence. Important bioluminescentcompounds for purposes of labeling are luciferin, luciferase andaequorin.

An antibody molecule of the present invention may be adapted forutilization in an immunometric assay, also known as a “two-site” or“sandwich” assay. In a typical immunometric assay, a quantity ofunlabeled antibody (or fragment of antibody) is bound to a solid supportor carrier and a quantity of detectably labeled soluble antibody isadded to permit detection and/or quantitation of the ternary complexformed between solid-phase antibody, antigen, and labeled antibody.

Typical, and preferred, immunometric assays include “forward” assays inwhich the antibody bound to the solid phase is first contacted with thesample being tested to extract the antigen from the sample by formationof a binary solid phase antibody-antigen complex. After a suitableincubation period, the solid support or carrier is washed to remove theresidue of the fluid sample, including unreacted antigen, if any, andthen contacted with the solution containing an unknown quantity oflabeled antibody (which functions as a “reporter molecule”). After asecond incubation period to permit the labeled antibody to complex withthe antigen bound to the solid support or carrier through the unlabeledantibody, the solid support or carrier is washed a second time to removethe unreacted labeled antibody.

In another type of “sandwich” assay, which may also be useful fordetection of CD48 according to the diagnostic method of the presentinvention, the so-called “simultaneous” and “reverse” assays are used. Asimultaneous assay involves a single incubation step as the antibodybound to the solid support or carrier and labeled antibody are bothadded to the sample being tested at the same time. After the incubationis completed, the solid support or carrier is washed to remove theresidue of fluid sample and uncomplexed labeled antibody. The presenceof labeled antibody associated with the solid support or carrier is thendetermined as it would be in a conventional “forward” sandwich assay.

In the “reverse” assay, stepwise addition first of a solution of labeledantibody to the fluid sample followed by the addition of unlabeledantibody bound to a solid support or carrier after a suitable incubationperiod is utilized. After a second incubation, the solid phase is washedin conventional fashion to free it of the residue of the sample beingtested and the solution of unreacted labeled antibody. The determinationof labeled antibody associated with a solid support or carrier is thendetermined as in the “simultaneous” and “forward” assays.

In another embodiment, said diagnostic method is effected at the mRNAlevel, in which case said determination comprises measuring the level ofCD48 mRNA expression by appropriate means, whereby elevated levels ofCD48 mRNA, in comparison with a negative control, indicates the presenceof an allergic condition, specifically, asthma.

Appropriate means for measuring mRNA levels include applying the methodsof RT-PCR, PCR, Nucleic acid based ELISA and hybridization with alabeled probe, e.g. in Northern blot, or in situ hybridization. Theseare well known methods which are familiar to the man skilled in the artof molecular biology.

Nucleic acid based ELISA may be performed by coating ELISA plates withantisense nucleic acid sequence derived from CD48, incubating tagged orlabeled RT-PCR products obtained from the examined sample with theplates and quantitating the bound nucleic acid sequence. Tag's suitablefor such purpose may be for example avidin/biotin, GFP, myc, FLAG andthe like.

In one further specific embodiment of the diagnostic method of theinvention, when effected at the mRNA level, said determination comprisesproviding primers for specific amplification of CD48 transcripts,together with nucleotides and amplification reagents, providingconditions for allowing CD48 amplification, whereby elevated levels ofCD48 amplification products, in comparison with a negative control,indicates the presence of at least one of asthma, allergy or an asthma-or allergy-related condition.

Thus, as used herein “RT-PCR” refers to a process of reversetranscription of mRNA into cDNA which is subsequently subjected to PCRreaction. PCR (Polymerase Chain Reaction) involves amplifying one ormore specific nucleic acid sequences by repeated rounds of synthesis anddenaturing under appropriate conditions.

PCR requires two primers that are capable of hybridization with asingle-strand of a double-stranded target nucleic acid sequence which isto be amplified under appropriate hybridization conditions. In PCR, thisdouble-stranded target sequence is denatured and one primer is annealedto each single-strand of the denatured target. The primers anneal to thetarget nucleic acid at sites removed (downstream or upstream) from oneanother and in orientations such that the extension product of oneprimer, when separated from its complement, can hybridize to theextension product generated from the other primer and target strand.Once a given primer hybridizes to the target sequence, the primer isextended by the action of a DNA polymerase. DNA polymerase which is heatstable is generally utilized so that new polymerase need not be addedafter each denaturation step. Such thermostable DNA polymerase is knownto one of ordinary skill in the art, e.g. Taq polymerase. The extensionproduct is then denatured from the target sequence, and the process isrepeated.

In a specifically preferred embodiment, the primer extension or PCRproduct may be un-labeled. In this case, the gel-banding pattern of theresulting fragments may be visualized by ethidium bromide (EtBr), or bysilver staining. Alternatively, the primer extension or PCR product maybe body-labeled, by using labeled nucleotide during the PCR reaction.The term “label” as used herein refers to any atom or molecule which canbe used to provide a detectable (preferably quantifiable) signal, andwhich can be attached to a nucleic acid or protein. Labels may providesignals detectable by fluorescence, radioactivity, colorimetry,gravimetry, X-ray diffraction or absorption, magnetism, enzymaticactivity, and the like.

As used herein, the term “nucleic acid” refers to polymer ofnucleotides, which may be either single- or double-stranded, which is apolynucleotide such as deoxyribonucleic acid (DNA), and, whereappropriate, ribonucleic acid (RNA). The terms should also be understoodto include, as equivalents, analogs of either RNA or DNA made fromnucleotide analogs, and, as applicable to the embodiment beingdescribed, single-stranded (such as sense or antisense) anddouble-stranded polynucleotides. The term DNA used herein alsoencompasses cDNA, i.e. complementary or copy DNA produced from an RNAtemplate by the action of reverse transcriptase (RNA-dependent DNApolymerase).

It is important to note that the results obtained through the hereindescribed diagnostic methods are always compared to a reference value,obtained e.g. from a negative control, which assists the medicalpractitioner to arrive at the correct diagnosis, positive or negative.

The inventors tested the involvement of key molecules which arehallmarks of mechanisms in the allergic asthmatic response such as e.g.STATE, IL-4, IL-13, IL-5 and eotaxin-1. These mechanisms have beenpreviously showed to regulate several genes that were present on the“asthma genome signature” such as TFF2, ADAM8, eotaxin-1 and arginase.Nevertheless, numerous genes related to allergic inflammation areSTATE-independent, including CXCL10, CXCL12 and Clq [Fulkerson, P. C. etal. (2004) J. Immunol. 173: 7565-74]. Mechanistic analysis of thesepathways revealed that CD48 was upregulated in the absence of STATE,IL-4 and IL-13. Interestingly, CD48 expression was found to bespontaneously upregulated in inducible IL-13 transgenic mice.

More particularly, to determine whether IL-3 physiologically upregulatesCD48 in allergic disorders, CD48 expression on eosinophils was evaluatedin a murine experimental asthma and allergic peritonitis. The dataobtained from the model presented herein demonstrate that CD48expression increased in a time-dependent fashion after allergenchallenge. IL-3 neutralization in OVA-challenged mice reduced eosinophilCD48 expression, but not to the baseline level that is observed insaline-treated mice. Thus, although IL-3 is the only identified cytokinethat up-regulates eosinophil CD48 expression, it is unlikely to be theonly factor responsible for this phenomenon in vivo. It is possible thatIL-3 and IL-4 act in concert to influence CD48 expression on variouscell types. Alternatively, higher doses of anti-IL-3 may be required fora more dramatic effect.

Cross-linking of CD48 on human eosinophils triggered EPO but notcytokine release even in the presence of IL-3. It is possible that IL-3potentiates the responses elicited by CD48. In support of thishypothesis, it is the inventors' observation that IL-3 enhances theability of eosinophils to internalize E. coli via CD48 (Munitz et al.,unpublished observations). Furthermore, IL-3 has been shown to primeeosinophils and augment eosinophil-LTC4 generation in response tocalcium ionophore and enhance cytotoxicity towards antibody-coatedhelminthes [Rothenberg, M. E. et al. (1988) J. Clin. Invest. 81:1986-1992]. Therefore, it is possible that CD48 together with IL-3regulates the release of specific mediators that are beneficial to thehost in helminth and bacterial infections (i.e. innate mechanisms) butunfavorable in allergic settings (i.e. adaptive mechanisms).

Thus, the inventors may hypothesize that compensatory mechanisms (yet tobe defined) present in the lungs of allergen-challenged mice canupregulate CD48 in the absence of one single pathway. It should be notedthat the possibility that CD48 is upregulated in the lungs ofOVA-challenged mice due to local inflammation and recruitment ofCD48-expressing cells, cannot be ruled out. Yet, upregulation of CD48was unchanged in STAT-6, IL-4, IL-13 and eotaxin-1/IL-5 deficient mice,which fail to develop an eosinophil infiltrate (data not shown). Thus,upon allergen challenge lung expression of CD48 remains unaltered. Thiscould result from compensatory mechanisms that recruit other CD48⁺ celltypes such as lymphocytes and neutrophils rather than eosinophils.

It is likely that CD48 signaling cascade contributes to eosinophilactivation and degranulation in asthma. As complex networks ofactivating and inhibitory signals govern the responses coordinated byeosinophils, increased CD48 expression might shift the resting thresholdof eosinophils toward activation.

While the role and pathways regulating NKT cell functions in allergicinflammations such as asthma are still to be determined, the results ofthe present invention suggests that 2B4 does not play a significant rolein their activation in allergic settings.

CD48 can induce signal transduction as it binds Lck, Fyn and G proteins[Patel, V. P. et al. (2001) J. Immunol. 166: 754-64; Stefanova, I, etal. (1991) Science 254: 1016-9)]. Cross-linking of CD48 on purifiedtonsillar B cells significantly increased CD40-mediated activation[Klyushnenkova, E. N. et al. (1996) Cell Immunol. 174: 90-8], andcross-linking CD48 in combination with IL-4 and/or IL-10 is able toinduce B cell aggregation, proliferation and IgG secretion. Therefore,the anti-CD48 agent used by the method of the invention may target otherdown-stream member molecules of the CD48 stimulatory pathway, such asLck, Fyn and G proteins.

More particularly, CD48 is upregulated on the single cell level by atleast two mediators that are expressed in the asthmatic milieu: IL-3 andIL-4. The abundance of pathways that regulate CD48 expression in vivohighlights CD48 importance. This led the inventors to investigate theCD48-CD2-2B4 axis in allergic eosinophilic airway inflammationpathogenesis. To examine this, the inventors administered anti-CD48,anti-CD2 and anti-2B4 neutralizing antibodies prior to allergenchallenge. Strikingly, neutralization of CD48 significantly reducedeosinophilic inflammation and cytokine expression (i.e Th2 cytokines;IL-5, IL-4 and IL-13, proinflammatory cytokines; TNF-α, chemokines;eotaxin-2) in the BALF. Moreover, it abrogated lung inflammation(alveolar space, perivascular and peribronchial) airway smooth musclethickening, epithelial shedding, goblet cell hyperplasia and mucusproduction. Neutralization of CD2 caused a ˜40-50% reduction in theseinflammatory parameters while anti-2B4 treated mice displayed nosignificant effect.

Several mechanisms could account for the anti-inflammatory effects ofblocking CD48. CD48 deficient mice show considerable defects in CD4⁺ Tcell activation. The inhibitory effect of anti-CD48 treatment in oursettings is likely to be only partially dependent on T cellco-stimulation via CD2, since anti-CD2 treated mice displayed a mildreduction of the disease parameters in comparison to anti-CD48 mAbtreated mice. Supporting this finding is the observation that CD2deficient mice do not display the same effects observed in CD48deficient mice. This suggests a broader and more substantial role forCD48 in the immune system than recognized. Several line of evidencesupports a broad immunological role for CD48.

The demonstration that CD48 is up-regulated in experimental asthmaindicates the role of this molecule in human disease as well.Interestingly, CD48 was up-regulated both in human peripheral bloodeosinophils and in human nasal polyp eosinophils of atopic asthmaticsvs. normal controls. The latter finding is of great consideration sincethe most prevalent disease associated with nasal polyposis is bronchialasthma.

The present results clearly define a novel pathway that is criticallyinvolved in the orchestration and regulation of experimental and humanasthma. Therefore, CD48 is herein presented as a new target for futuretherapeutic and diagnostic approaches.

As referred to herein, the term “asthma” or “asthmatic condition” refersto a medical condition characterized by recurrent attacks of paroxysmaldyspnea, with airway inflammation and wheezing due to spasmodiccontraction of the bronchi. In other words, asthma is an inflammatorycondition of the bronchial airways, characterized by airflow obstructionand bronchial hyper-responsiveness, resulting in increased mucusproduction, mucosal swelling and muscle contraction. These changesproduce airway obstruction, chest tightness, coughing and wheezing. Whensevere, this can cause severe shortness of breath and low blood oxygen.

The asthmatic condition has various etiologies, including allergicmanifestations in sensitized individuals (“allergic asthma”), asthmaprovoked by factors such as vigorous exercise, irritant particles, viralrespiratory infections and/or psychological stress (“bronchial asthma”or “spasmodic asthma”), amongst others.

Allergy may be defined as a reaction to foreign substances (allergens)by the immune system. Examples of allergens include pollens, dust mite,molds, dander, and certain foods.

The most common allergic conditions include hay fever (allergicrhinitis), asthma, allergic eyes (allergic conjunctivitis), allergiceczema, hives (urticaria), and allergic shock (also called anaphylaxisand anaphylactic shock).

More specifically, “conjunctivitis” is an inflammation of theconjunctiva (the outermost layer of the eye and the inner surface of theeyelids), most commonly due to an allergic reaction or an infection(usually bacterial or viral).

“Atopic dermatitis” (AD) is a chronic, highly pruritic, eczematous skindisease that follows patients from early childhood into puberty andsometimes adulthood. Also referred to as eczematous dermatitis, thedisease often has a remitting/flaring course, which may be exacerbatedby social, environmental, and biological triggers.

For the method of treatment provided in the present invention, saidtherapeutic effective amount, or dosage, is dependent on severity andresponsiveness of the disease state to be treated, with the course oftreatment lasting from several days to several months, or until a cureis effected or a diminution of the disease state is achieved. Optimaldosing schedules can be calculated from measurements of drugaccumulation in the body of the patient. Persons of ordinary skill caneasily determine optimum dosages, dosing methodologies and repetitionrates. In general, dosage is calculated according to body weight, andmay be given once or more daily, weekly, monthly or yearly, or even onceevery 2 to 20 years. Persons of ordinary skill in the art can easilyestimate repetition rates for dosing based on measured residence timesand concentrations of the anti-CD48 agent in bodily fluids or tissues.Following successful treatment, it may be desirable to have the patientundergo maintenance therapy to prevent the recurrence of the diseasestate, wherein the anti-CD48 agent is administered in maintenance doses,once or more daily.

Various methods of administration may be used for delivering theanti-CD48 agent to a subject in need. It should be emphasized that alocalized administration of the anti-CD48 agent is specificallypreferred. Said agent may be delivered via intravenous (i.v.),intramuscular (i.m.), intraperitoneal (i.p.) injections, orally (inliquid form or prepared as dosage unit forms like capsules, pills,lozenges, etc.). Alternatively and preferably, the anti-CD48 agent mayalso be delivered in a local manner, via transdermal delivery usingpatches, ointment or cream. Other routes of administration areintranasal, intradermal, sub-lingual, and intrathecal, although systemicdelivery may be also used. Possible delivery devices include aerosol(inhalation), liposomal carriers, drops, ready-to-use syringes, pillsand capsules, amongst others.

Taken together the present results suggest that CD48 may serve as amultifaceted molecule that regulates several eosinophil effectorfunctions in disease settings. For example, elevated levels of CD48 oneosinophils and basophils correlated with increased infiltration ofthese cells to the lung, BALF and spleen. In addition, CD48 has beenreported to function as an adhesion molecule [Yokoyama, S. D. et al.(1991) J. Immunol. 146: 2192-2200], and it can bind directly to heparinsulfate on the surface of epithelial cells [Ianelli, C. J. et al. (1998)J. Biol. Chem. 273: 23367-23375]. Consequently, CD48 may influencehoming, transmigration, and tissue retention of eosinophils in allergicsettings.

Therefore, the present invention also provides the use of an anti-CD48agent as a preventive vehicle for allergic conditions. Given the presentfindings that CD48 is one of the molecules at the top of the signalingcascade involving the allergic response (which triggers the asthmaattack), inhibiting CD48 function/expression is a way of preventing theasthmatic or allergic condition to take place. Such a drug is certainlya leap forward in asthma and allergy drug development, in view, thatasthmatic and/or allergic patients are usually treated with drugs thatease the symptoms, but are not spared from the agony of the attack. Apreventive drug is hence much in demand for this target population.

By “patient” or “subject in need” it is meant any mammal who may beaffected by the above-mentioned conditions, and to whom the treatmentand diagnosis methods herein described is desired, including human,bovine, equine, canine, murine and feline subjects. Preferably saidpatient is a human.

In conclusion, the present results support the idea that CD48 has animportant role in eosinophil activation in a variety of allergicconditions not previously described.

Lastly, the present invention also provides a kit for the diagnosis ofan allergic condition, specifically, asthma and related conditions, saidkit comprising as follows:

-   -   (a) an agent for determining the presence of an analyte of        interest, wherein said analyte is selected from the group        consisting of: CD48 protein and CD48 mRNA;    -   (b) calibration means; and    -   optionally a manual of instructions of how to perform the        diagnostic test.

Where the analyte is CD48 protein, said agent is an anti-CD48 antibody.Said antibody may be conjugated to a detectable label or not. In casesaid antibody is not conjugated to a detectable label, a second antibodyis also supplied by the kit of the invention, wherein said secondantibody is conjugated to a detectable label, it is produced in adifferent species from the first antibody, and it is capable ofdetecting the first antibody.

Evidently, where the analyte is CD48 mRNA, said agent may be aCD48-specific probe, said probe being a oligonucleotide sequencecomplementary and specific to the CD48 mRNA sequence, labeled by anysuitable means, e.g. immunofluorescence labeling, luminescence,radioisotopes, etc.

Alternatively, the detection of CD48 mRNA may be effectuated throughamplification of said specific mRNA, in PCR or RT-PCR reactions, and inwhich case said agent is a CD48-specific primer (typically specificcomplementary oligonucleotides).

The detection per se may be performed e.g. using FACS analysis, whereinthe sample is analyzed by FACS using the appropriate parameters andcomparing to a negative control. Alternatively, the cells obtained fromthe collected sample may be fixed onto a slide, and CD48 expressiondetected via anti-CD48 antibodies or labeled probes (in situ detection).Yet another detection method which may be utilized involves protein orRNA extraction and analysis, via e.g. Western blot, ELISA, Northern blotor PCR. Any one of these alternatives is specified in the manual ofinstructions accompanying the kit, accordingly.

As mentioned herein, calibration means may be a sample of a negativecontrol in order to give a yes/no binary answer. The calibration mayalso comprise a full calibration curve in order to give a quantitativeanswer as regards the severity of the allergic reaction, or of theasthmatic condition.

In another embodiment, said kit may optionally further comprise at leastone of the following components: means for obtaining a blood sample,means for isolating leucocytes from the blood sample, and means fordetecting the anti-CD48 antibody or CD48-specific probe.

The invention further provides a pharmaceutical composition for thetreatment of an allergic condition comprising as an active ingredient atherapeutically effective amount of an anti-CD48 agent and optionallyfurther comprising pharmaceutically acceptable carrier, diluent,excipients and/or additive.

The present invention is defined by the claims, the contents of whichare to be read as included within the disclosure of the specification,and will now be described by way of example with reference to theaccompanying Figures.

Disclosed and described, it is to be understood that this invention isnot limited to the particular examples, process steps, and materialsdisclosed herein as such process steps and materials may vary somewhat.It is also to be understood that the terminology used herein is used forthe purpose of describing particular embodiments only and not intendedto be limiting since the scope of the present invention will be limitedonly by the appended claims and equivalents thereof.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a”, “an” and “the” include plural referentsunless the content clearly dictates otherwise.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.

The following Examples are representative of techniques employed by theinventors in carrying out aspects of the present invention. It should beappreciated that while these techniques are exemplary of preferredembodiments for the practice of the invention, those of skill in theart, in light of the present disclosure, will recognize that numerousmodifications can be made without departing from the spirit and intendedscope of the invention.

EXAMPLES Experimental Procedures

A number of methods of the art of molecular biology are not detailedherein, as they are well known to the person of skill in the art. Suchmethods include site-directed mutagenesis, PCR cloning, expression ofcDNAs, analysis of recombinant proteins or peptides, transformation ofbacterial and yeast cells, transfection of mammalian cells, and thelike. Textbooks describing such methods are e.g., Sambrook et al.,Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory;ISBN: 0879693096, 1989, Current Protocols in Molecular Biology, by F. M.Ausubel, ISBN: 047150338X, John Wiley & Sons, Inc. 1988, and ShortProtocols in Molecular Biology, by F. M. Ausubel et al. (eds.) 3rd ed.John Wiley & Sons; ISBN: 0471137812, 1995. These publications areincorporated herein in their entirety by reference. Furthermore, anumber of immunological techniques are not in each instance describedherein in detail, as they are well known to the person of skill in theart. See e.g., Current Protocols in Immunology, Coligan et al. (eds),John Wiley & Sons. Inc., New York, N.Y., 1999.

Reagents and Chemicals:

All chemicals used in this study were purchased from Sigma (Rehovot,Israel) and were of the best available grade.

Antibodies

FITC conjugated anti-CCR3 was obtained from R&D Systems (Minneapolis,Minn.). Anti-CD3-APC, anti-VLA2-PE (DX5), anti-CD4-PE-Cy5, anti-rat-PE,anti-rat-FITC, streptavidin-PE and streptavidin-Cy5 were all purchasedfrom eBioscience, (San Diego, Calif.). Anti-B220-APC, anti-CD2 andanti-CD48-PE were obtained from Biolegend (San Diego, Calif.). Anti-2B4mAb (a kind gift of Dr. Vinay Kumar, University of Chicago) and anti-CD2were conjugated to biotin using a standard protocol.

Mice:

Balb/c mice were obtained from the National Cancer Institute (Frederick,Md.) or Harlan laboratories (Israel) and housed under specificpathogen-free conditions. Mice deficient in STAT6 or IL-4Rα in theBalb/c background were obtained from Jackson Laboratories (Bar Harbor,Me.). IL-13-deficient mice and mice deficient in both IL-4 and IL-13were kindly provided by Dr. Andrew McKenzie. Generation ofeotaxin-deficient IL-5-transgenic mice was previously described [Mishra,A. et al. (1999) J. Clin. Invest. 103(12):1719-27] Mice carrying thetetracycline inducible IL-13 transgene under the regulation of the Claracell 10 (CC10) lung promoter have been previously described [Horejsi, V.et al. (1999) Immunol. Today 20: 356-361].

Experimental Asthma:

BALB/c female mice (7-8 weeks old) were obtained from HarlanLaboratories (Israel) and housed under specific pathogen-freeconditions. Mice were sensitized by i.p. injection with 100 μg of OVAadsorbed onto 1 mg of aluminum hydroxide in 250 μl of saline on days 0and 14. On days 24 and 27, the mice were lightly anesthetized withinhaled isofluorane and challenged intranasally with 50 μg of OVA orsaline. The allergen challenge was performed by applying 50 μl of thesame to the nostrils using a micropipette with the mouse held in asupine position. After instillation, the mice were held upright untilalert. Mice were sacrificed by isofluorane inhalation at the indicatedtime points (0-24 hrs) following allergen challenge and BALF wasperformed for differential cell counts [Van Rijt, L. S. et al. (2004) J.Immunol. Meth. 288: 111-121]. In addition, lungs were excised, digestedas described [Southam, D. S. et al. (2005) J. Allergy Clin. Immunol.115: 95-102], and differential cell count performed.

In neutralization experiments, anti-IL-3 (clone 8F8) (2 mg/mouse in 300μl saline), anti-CD48, anti-CD2 (Biolegend, San Diego, Calif.),anti-2B4, or appropriate isotype matched controls (Hamster IgG and RatIgG) were administered i.p on day 23 (24 hrs prior to allergenchallenge) and on days 24 and 27 one hour prior to allergen challenge(250 μg/mouse in 300 μl saline). These concentrations were chosenbecause they had been previously shown to have a neutralizing effect invivo. Mice were sacrificed 18 hrs after the last allergen challenge.BALF was performed for differential cell counts and eosinophils wereassessed for CD48 expression. In addition, lungs were excised, fixed in4% paraformaldehyde, paraffin embedded and stained by H&E(hematoxylin/eosin). Calculation of total lung inflammation wasperformed by assessing alveolar space and perivascular and peribronchialinfiltrate using the following key: O-no inflammation, 1-lightinflammation, 2-moderate inflammation, and 3-severe inflammation.

Experimental Allergic Peritonitis:

BALB/c female mice (8-10 weeks old) were sensitized subcutaneously ondays 0 and 7 with 100 μg of OVA adsorbed onto 1.6 mg of aluminumhydroxide in 300 μl saline. On day 11, the mice were challenged i.p.with 3 μg of OVA in 200 μl of saline and sacrificed at the indicatedtime points (6 hrs-48 hrs). Thereafter, the peritoneal cavity was washedwith 5 ml of Tyrode's gelatin buffer for differential cell counts.

For experiments involving IL-5 transgenic mice, mice were obtained asdescribed [Finkelman, F. D. et al. (1993) J. Immunol. 151: 1235-1244].

All experiments involving animals and primary animal cells were approvedby the Institutional Animal Experimentation Ethics Committee of theHadassah Ein Kerem Hospital.

IL-3 Administration

IL-3 (Peprotech, Rocky Hill, N.J.) was administered intranasally orsystemically in lightly anesthetized (isofluorane) BALB/c female mice(7-8 weeks old). Briefly, recombinant murine IL-3 (2-4 μg in 50 μlsaline for intranasal administration and 8-10 μg in 100 μl saline) wasdelivered in conjunction with anti-IL-3 mAb (4-20 μg) (IL-3C). Thisco-injection triggers the formation of an IL-3/anti-IL-3 mAb complex(IL-3C) that slowly releases IL-3 with an in vivo half-life of ˜24 hrs,as compared to a half-life of several minutes for free IL-3 [Finkelman(1993) ibid.]. The mice received IL-3C every other day for 21 days. Micewere sacrificed 24 hrs after the last administration of IL-3C. Spleen,lung and BALF cells were assessed for CD48 expression by FACS (seebelow).

Microarray Hybridization and Analysis:

Microarray hybridization was performed by the Affymetrix Gene Chip Corefacility at Cincinnati Children's Hospital Medical Center (Cincinnati,Ohio, USA), as previously described [Bochner, B. S. (2004) J. AllergyClin. Immunol. 113:3-9]. Briefly, total lung RNA was obtained usingTrizol (Invitrogen, Grand Island, N.Y.) according to the manufacturers'instructions. RNA was converted to cDNA with Superscript choice for cDNAsynthesis (Invitrogen, Carlsbad, Calif., USA) and subsequently convertedto biotinylated cRNA with Enzo BioArray™ High Yield™ RNA Transcriptlabeling kit (Enzo Diagnostics, Farmingdale, N.Y.). After hybridizationto the murine U74Av2 GeneChip (Affymetrix, Santa Clara, Calif., USA),the gene chips were automatically washed and stained with 10streptavidin-phycoerythrin by using a fluidics system. The chips werescanned with a Hewlett Packard Gene Array Scanner. This analysis wasperformed with one mouse per chip (n>3 for each allergen challengecondition and n>2 for each saline challenge condition). From data imagefiles, gene transcript levels were determined using algorithms in theMicroarray Analysis Suite Version 4 software (Affymetrix). Differencesbetween saline- and OVA-treated mice were also determined using theGeneSpring software (Silicon Genetics, Redwood City, Calif., USA). Datafor each allergen challenge time point was normalized to the average ofthe saline-treated mice.

Northern Blot Analysis:

Total lung RNA (10-20 μg) was analyzed by electrophoresis in anagarose-formaldehyde gel, transferred to Gene Screen transfer membranes(NEN, Boston, Mass., USA) in 10×SSC and cross-linked by UV radiation.Sequence confirmed probes were obtained from American Type TissueCulture Collection (ATCC, Rockville, Md., USA) and labeled with ³²Pusing the Klenow reaction and random priming. Blots were hybridizedunder standard conditions.

Human Eosinophil Purification:

Eosinophils were purified from peripheral blood of atopic asthmatics(see below) or age and sex matched normal individuals (blood eosinophillevels 5-10%) by MACS negative immunomagnetic separation as previouslydescribed [Munitz, A. et al. (2005) ibid. J. Immunol. 174: 110-118].Asthmatic donors were all atopic individuals (total IgE>100 IU/ml blood)requiring intermittent 132-agonist treatment (FEV1 values ranging from75%-90% of normal). Non-asthmatic gender-matched controls werenon-atopic and had FEV1 values greater than 95% of normal.

Written informed consent was obtained from all volunteers according tothe guidelines established by the Hadassah-Hebrew University HumanExperimentation Helsinki Committee.

Briefly, venous blood (50-100 ml) was collected in heparinized syringesand left to sediment in 6% dextran (Amersham Biosciences, Uppsala,Sweden). Leukocytes were centrifuged on Ficoll-Hypaque (density 1.077,Amersham Biosciences, Uppsala, Sweden), for 25 min, 700 g, 22° C.Neutrophils and contaminating lymphocytes were tagged in thegranulocyte-enriched pellet with micromagnetic beads bound to anti-CD16and anti-CD3 antibodies (Miltenyi Biotech, GmbH, Bergisch Gladbach,Germany). Eosinophils were purified by passing the cell suspensionthrough a magnetic column (MACS). They were collected at a purity of atleast 98%, according to Kimura's staining, and at a viability of atleast 98%, as evaluated by trypan blue staining. Eosinophil preparationswere re-suspended in medium containing RPMI-1640, 200 U/ml penicillin,200 μg/ml streptomycin and 10% v/v heat inactivated FCS (RPMI 10%)

Human Nasal Polyp Digestion:

Cells were isolated and obtained from nasal polyps of atopic asthmaticpatients [Munitz, A. et al. (2005) Blood October 27; Epub ahead ofprint] or age and sex matched non-asthmatic individuals according toguidelines established by the Hadassah-Hebrew University HumanExperimentation Helsinki Committee. Nasal polyps were washed twice inRPMI-2% FCS, minced to fragments of ˜1 mm³ and subsequently digested byincubation for 60 min at 37° C. with an enzyme cocktail containingcollagenase type-I (6 mg/gram tissue), hyaluronidase (3 mg/gram tissue),and DNase (100 μg/gram tissue). The digested tissue was filtered througha 150 mesh nylon cloth. Collected cells contained >55-90% eosinophils(Kimura's staining) and had a viability of >94% (Trypan blue exclusion).Contaminating cells were usually macrophages and to a lesser extent,lymphocytes. Eosinophils in the cell suspension were identified asSSC^(high) and CCR3⁺ cells using FACS analysis.

Asthmatic donors were all atopic individuals (total IgE>100 units)requiring intermittent β₂-agonist treatment (FEV₁ values ranging 75%-90%of normal FEV₁). Non asthmatic gender-matched controls were non-atopicand had FEV₁ values greater than 95% of normal.

Human Eosinophil Cell Culture:

For receptor up-regulation experiments, freshly isolated humanperipheral blood eosinophils were seeded in 96 plate U-shaped wells(Nunc, Roskilde, Denmark) (2×10⁵/200 μl) in RPMI-10%, and incubated (37°C., 5% CO₂) for the indicated time points with IL-3 or with variousother cytokines or chemokines (2-200 ng/ml, all purchased fromPeprotech, Rocky Hill, N.J.). Thereafter, the cells were washed and CD48expression was assessed by FACS.

For mediator release assays, 96-well plates (Nunc, Roskilde, Denmark)were pre-coated with sheep anti-mouse IgG F(ab) (25 μg/ml) in PBS, fortwo hours, at 37° C., 5% CO₂. Afterwards plates were washed three timeswith PBS and incubated with anti-CD48 mAb (Pharmingen, San Diego,Calif.) or an irrelevant isotype-matched control mAb (DAKO, Denmark) (1μg/ml, 2 hrs at 37°, 5% CO₂) and washed three times. Freshly isolatedeosinophils were seeded in these pre-coated wells (2×105/200 μl) inRPMI-10% (as described above) and incubated for 30 min-18 hrs (37° C.,5% CO₂). At the end of the incubation, cells were centrifuged (300 g, 5min, 4° C.), supernatants collected, aliquoted, and stored at −80° C.until assessed for EPO (eosinophil peroxidase) activity.

Eosinophil Peroxidase (EPO) Determination;

EPO release was determined by a colorimetric assay. Briefly, eosinophilculture supernatants (50 μl) were incubated (10-15 min, 37° C., 5% CO₂)with a substrate solution that contained 0.1 mM O-phenylenediaminedihydrochloride in 0.05 M Tris buffer (pH 8.0), 0.1% Triton X-100 (37°C., 5% CO₂) and 1 mM hydrogen peroxide (Merck, Darmstadt, Germany). Thereaction was stopped by the addition of 4 mM sulfuric acid (BDH, Dorset,UK) and the absorbance was determined at 492 nm in a spectrophotometer(PowerWave™ XS, Bio-Tek Instruments, Bad Friedrichshall, Germany).

Flow Cytometry:

For assessment of CD48 expression on human eosinophils, cells (2×10⁵)were incubated with anti-CD48 (1 μg/ml, clone 4H9, Santa Cruz, Calif.,USA) a final volume of 1000 of Hanks Balanced Salt Solution supplementedwith 0.1% bovine serum albumin and 0.02% sodium azide (HBA) for 30 minon ice. Thereafter the cells were washed and goat anti-mouse FITC(1:500, Jackson, Immunoresearch Laboratories, West Grove, Pa., USA) wasadded. After 30 min incubation (30 min, 37° C., 5% CO₂), the cells werewashed and analyzed on a Beckton Dickinson FACScalibur™ System (BecktonDickinson, San Jose, Calif., USA).

Total BALF cells (2×10⁵) of treated mice were incubated with theaforementioned antibodies in a final volume of 100 μl of Hanks BalancedSalt Solution supplemented with 0.1% bovine serum albumin and 0.02%sodium azide for 30 min on ice. Cell staining was performed byfour-color flow cytometry using anti-CD3 APC, anti-c-kit Pe-Cy5,Anti-FcεRI FITC, anti-CD4 Pe-Cy5, anti-CD48 PE (eBioscience, San Diego,Calif., USA), and anti-CCR3 FITC (R&D, Systems, Minneapolis, Minn.,USA). Thereafter, differential cell populations were electronicallygated and assessed for expression of CD48, CD2 or 2B4. For FACSanalysis, differential cell populations were defined as follows:Eosinophils—SSC^(high), CCR3^(high), CD49d^(high), c-kit^(low),FcεRI^(low), Ly49b⁻ and CD3⁻; Neutrophils—SSC^(high), CCR3⁻ and CD3⁻;Lymphocytes—SSC^(low), CCR3⁻ and either CD3⁺ or B220⁺;Mononcytes/macrophages—SSC^(high), FSC^(high), CCR3⁻, CD3⁻ and FcεRI⁻;NK cells—SSC^(low), CCR3⁻, DX5⁺ and CD3⁻: NKT cells SSC^(low), CCR3⁻,DX5⁺ and CD3⁺. The different cell types were identified through theirsurface antigens and physical parameters (SSC vs. FSC) as previouslydescribed [Van Rijt, L. S. et al. (2004) J. Immunol. Methods 288:111-12]. For each staining at least ten thousand cells were collectedand data analysis was performed using CellQuest™ software (Mansfield,Mass., USA).

Mediator Assessment:

Cytokines were measured with kits purchased from the following sources:IL-5; e-bioscience (San Diego, Calif.), IL-4 and IL-13; Biolegend (SanDiego, Calif.), Eotaxin-2 and TNF-α; R&D systems ((Minneapolis, Minn.).ELISA procedures were carried out according to the manufacturers'instructions. Lower detection limits for the various assays were: 7.8pg/ml, 2 pg/ml, 16 pg/ml, 32 pg/ml and 16 pg/ml respectively. Assessmentof RANTES, MCP-1 and MCP-5 was performed using the RayBio® cytokineprotein Arrays (RayBiotech, Norcross, Ga., USA), according tomanufacturer's instructions.

Quantification of Lung Inflammation:

Histological studies were performed as follows; the right upper lobe ofsaline or allergen-challenged lungs was fixed in 3.7% paraformaldehyde,embedded in paraffin, deparaffinized and stained with hematoxylin andeosin or periodic acid Schiff.

Inflammatory Score:

H&E stained slides were examined by two blinded observers and gradedusing a standard scoring method (0=normal; 1=mild; 2=intermediate;3=severe inflammation). At least three medium sized bronchioles andblood vessels were examined per slide.

PAS Staining Quantification:

To quantify the level of mucus expression in the airway, the number ofPAS-positive and PAS-negative epithelial cells in individual bronchioleswas counted. At least three medium-sized bronchioles (defined by havingapproximately 90.150 luminal airway epithelial cells) were counted perslide. Results are expressed as the percentage of PAS-positive cells perbronchiole, which is calculated from the number of PAS-positiveepithelial cells per bronchus divided by the total number of epithelialcells of each bronchiole.

Peribronchial Smooth Muscle Thickness Quantification:

To quantify the levels of peribronchial smooth muscle thickening, H&Estained slides were microphotographed and the mean pixel count of atleast 10 different positions per bronchiole of each smooth muscle layerwas obtained. This procedure was calculated from at least threemedium-sized bronchioles per mouse.

Reagents and Chemicals:

RPMI-1640 supplemented with L-glutamine, heat-inactivated Fetal CalfSerum (FCS) and penicillin-streptomycin solutions were obtained fromBiological Industries (Beit Haemek, Israel). All the chemicals used inthis study were purchased from Sigma (Rehovot, Israel) and were of bestavailable grade.

Statistical Analysis:

Statistical significance was calculated using parametric analysis(ANOVA, followed by students' t-test assuming equal variance). Valueswere considered significant at p<0.05.

Example 1

DNA Microarray Analysis Identifies CD48 as an Allergen-Induced Gene inAllergic Eosinophilic Airway Inflammation

Quantitative microarray analysis revealed that CD48 but not CD2 or 2B4mRNA expression was significantly increased in both the OVA- andAspergillus-induced allergic eosinophilic airway inflammation models(FIG. 1A-B respectively. In the OVA-induced experimental asthma model,kinetic analysis revealed that CD48 mRNA was significantly up-regulated18 hrs after the second allergen challenge but not after a singleallergen challenge or 3 hrs after the second allergen challenge (notshown). In the Aspergillus-induced experimental asthma model, CD48 wasup-regulated 18 hrs after the ninth allergen challenge. Subsequently,this data was confirmed by Northern Blot analysis. As shown in FIG. 1Cthere is a low basal expression of CD48 in the lungs of saline treatedmice. However, the levels of CD48 were significantly up-regulated afterOVA and Aspergillus challenge.

Example 2 Peripheral Blood Eosinophils and Nasal Polyp Eosinophils ofAtopic Asthmatics Express Increased Levels of CD48

The inventors examined whether CD48 expression on eosinophils iselevated in atopic asthmatic donors compared to normal controls. Asassessed by FACS analysis, peripheral blood eosinophils from atopicasthmatic donors expressed higher levels of CD48 (MFI 16.87±6.16, n=7,p<0.01) compared with eosinophils from non-asthmatic donors (MFI7.07±2.63) (FIG. 2A). Nasal polyposis has been linked to bronchialasthma and the percentage of infiltrating eosinophils in the polyps canreach as high as 60% [Eliashar, R. and Levi-Schaffer (2005) Curr. Opin.Otolaryngol. Head Neck Surg. 13: 171-175]. Nasal polyp eosinophilsobtained from asthmatic donors demonstrated significantly higher CD48levels (MFI 10.11±4.26, n=11, p<0.01) than nasal polyp eosinophils fromnon-asthmatic individuals (MFI 4.68±2.57) (FIG. 2B).

Example 3 The Expression of CD48 on Human Eosinophils is Up-Regulated byIL-3

The observation that human eosinophils from asthmatic donors displayelevated levels of CD48 suggests that its expression may be regulated bya mediator involved in asthma pathogenesis. To clarify which mediatormay regulate CD48, freshly isolated eosinophils were incubated withcytokines, growth factors and chemokines, including IL-2, IL-3, IL-4,IL-5, IL-8, IL-13, IFN-γ, GM-CSF, SCF, TGF-13, eotaxin-1, RANTES andMIP-1α that are found in the asthmatic milieu. Although IL-3, IL-5 andGM-CSF share a common β chain (βc) that transduces their signal, onlyIL-3 up-regulated CD48 expression (FIG. 3A). IL-3 elicited its effect ina concentration-dependent fashion, with a maximal effect at 20 ng/ml(1.51±0.13 fold increase, 2.11±0.13 fold increase and 1.91±0.06 foldincrease, respectively, following stimulation with 2, 20 or 200 ng/ml ofIL-3, n=5, p<0.001). In addition, kinetic analysis revealed thatIL-3-induced up-regulation peaked at 24 hours (2.14±0.15 fold increase,n=3, p<0.01) (FIG. 3B).

Example 4 Northern Blot Analysis Indicates that CD48 Expression isIndependent of STAT6, IL-4 and IL-13

The demonstration that CD48 has an inducible expression pattern oneosinophils during induction of experimental asthma indicates that thereis a factor in the asthmatic milieu that regulates this phenomenon.Asthma is a The associated process, therefore the inventors aimed todetermine whether signaling pathways such as STAT6 or cytokines such asIL-4 and IL-13 that are key regulators of the asthmatic response areinvolved in the up-regulation of CD48. Therefore, OVA- andAspergillus-induced experimental allergic-eosinophilic airwayinflammation protocols were employed to STAT6-, IL-13- and IL-4/IL-13deficient mice. Thereafter, total lung mRNA was extracted and subjectedto Northern blot analysis. As shown in FIG. 4A-D, the up-regulation ofCD48 expression was found to be independent of all of these factors inboth the OVA- and Aspergillus-induced models.

Nevertheless, inducible IL-13 transgenic mice display elevated levels ofCD48 starting after 6 days of IL-13 induction, indicating that IL-13over-expression is sufficient for CD48 overexpression (FIG. 4E).

Example 5 CD48 Activates Human Eosinophils to Release EPO

Expression of CD48 on the eosinophil surface suggests that eosinophilresponses may be regulated by this receptor. Cross-linking of CD48 onhuman eosinophils induced EPO release (FIG. 5). However, CD48cross-linking did not induce cytokine release, as IL-4, IL-8 and IFN-γwere not detected in the culture supernatants. Furthermore,cross-linking of CD48 in the presence of IL-3 did not enhance EPOrelease or cause cytokine release (data not shown).

Example 6 IL-3 Regulates CD48 Expression in Mice

Important effector mechanisms are likely to display conserved regulatorypathways between different species. Thus, the inventors verified whetherIL-3 up-regulated CD48 expression in vivo in the mouse. Intranasaladministration of IL-3 to BALB/c mice for 21 days significantlyincreased eosinophil, basophil and lymphocyte infiltration to the BALFand lungs compared with control (saline administration, FIG. 6A-B).Furthermore, IL-3 specifically up-regulated CD48 expression on BALF andlung eosinophils and basophils, but did not on lymphocytes, neutrophilsor macrophages (FIG. 6C-D). Consistent with this, intravenousadministration of IL-3C increased eosinophil and basophil numbers, aswell as their CD48 expression in the spleen (FIG. 6E-F).

In addition, as assessed by an in vivo cytokine capture assay[Finkelman, F. D. and Morris, S. C. (1999) Int. Immunol. 11: 1811-1818],systemic administration of IL-3C increased IL-4 production by 20-30 fold(data not shown). Thus, IL-3 activates mediator release in vivo.

In order to establish whether IL-3 is specifically responsible for CD48up-regulation in vivo, the expression of CD48 was examined ineosinophils from IL-5 transgenic mice in comparison to wild type mice.As shown in FIG. 6E, eosinophils from IL-5 transgenic mice displayedcomparable levels of CD48 compared to wild type mice (FIG. 6G).Therefore, in vivo up-regulation of CD48 expression on mouseeosinophils, like in vitro up-regulation of CD48 on human eosinophils,is induced by IL-3 but not IL-5 in vivo.

Example 7 CD48 is Up-Regulated on Murine Eosinophils in ExperimentalAsthma and Experimental Allergic Peritonitis

Two independent experimental allergy models were examined as to whetherCD48 is up-regulated in allergic conditions in mice: in vivoantigen-induced allergic airway inflammation (experimental asthma) andantigen-induced allergic peritonitis. In experimental asthma induced byOVA challenge, expression of CD48 by BALF eosinophils was significantlyup-regulated in a time-dependent fashion, while saline challenge had noeffect (FIG. 7A). The kinetics of CD48 expression was similar in theBALF and the lungs increasing 6 hrs after the last allergen challengeand peaking at 24 hrs (data not shown). Eosinophil CD48 expression wasalso increased in allergic peritonitis, increasing at 8 hrs and peakingat 48 hrs (FIG. 7B).

Example 8 Neutralization of IL-3 in Experimental Asthma Reduces CD48Expression

To determine whether IL-3 is responsible for the elevated expression ofCD48 observed in murine experimental asthma, neutralizing antibodies toIL-3 or isotype matched control antibodies were administered to OVAchallenged mice. Neutralization of IL-3 in OVA-challenged mice resultedin a 33% decrease (p<0.05) in CD48 expression by BALF eosinophils (FIG.8A), decreased the number of infiltrating BALF eosinophils (FIG. 8B) andattenuated lung inflammation (FIG. 8C) while an isotype-matched controlantibody had no effect. In addition, neutralization of IL-3 decreasedthe levels of IL-4 in the BALF of OVA-challenged mice from 79±2.7 pg/mlto 61±4.5 pg/ml, p<0.05, n=2 (data not shown).

Example 9 Cellular Source of CD48, CD2 and 2B4 in the Lungs

The inventors next determined the cellular source of CD48 and itsligands. Most of the cells in the lung and BALF expressed CD48; however,eosinophils expressed the highest levels of CD48 and were the maincellular source for its expression comprising ˜50-65% of CD48+ cells inthe lung (FIG. 9). Interestingly, murine eosinophils did not express 2B4and 2B4 expression was restricted to NK and NKT cells. In addition, CD2expression was limited to NKT, NK and CD4⁺ T cells (FIG. 9).

Example 10 Neutralization of CD48 Attenuates Eosinophilic Inflammation,Th2 and Proinflammatory Cytokines Expression in the BALF

The demonstration that CD48 is upregulated in allergic-eosinophilicairway inflammation raised the possibility that this type ofinflammation is dependent on CD48 and its ligands. Consequently, theinventors used specific neutralizing antibodies for CD48, CD2 and 2B4 toinvestigate their roles in this experimental regime (FIG. 10A-F).

Anti-CD48 mAb treatment prior to allergen challenge considerably reducedBALF inflammation. For example, eosinophilic inflammation wassignificantly decreased upon CD48 pretreatment (˜85%). Interestingly,anti-CD2 mAb pretreatment inhibited BALF inflammation to a lesser extentand caused a ˜45% reduction in BALF eosinophils. Pretreatment withanti-2B4 mAb did not alter eosinophilic inflammation (FIG. 10A). Inaddition, OVA-challenged mice displayed increased IL-4, IL-5, IL-13,TNF-α and eotaxin-2 levels (FIG. 10B-F). However, mice pretreated withanti-CD48 mAb showed a pronounced reduction of these cytokines (75%-93%decrease). In contrast, mice treated with anti-CD2 or anti-2B4 mAbexhibited only a ˜40-50% decrease in the BALF cytokine profile or had noeffect, respectively. Notably, all of the aforementioned effects werespecific since mice that were treated with control antibodies displayedequivalent cytokine levels to OVA-challenged mice.

The aforementioned antibodies were also analyzed by us to role out thepossibility that they may deplete targeted cells in vivo. As shown byFIG. 10G, administration of anti-CD48, anti-CD2 and anti-2B4 Abs did notalter splenic and peripheral blood cellular composition or numbers.

Example 11 Neutralization of CD48 Attenuates Lung Inflammation

These findings indicate that CD48 has a significant role in theallergen-induced inflammatory response. Accordingly, lung histologyfollowed by quantitative analysis was performed to assess the effects ofCD48 and its ligands on several parameters of lung inflammation (FIG.11A-D). As shown, OVA challenged mice, displayed evident perivascularand peribronchial eosinophilic inflammation, epithelial damage andairway muscle thickening. Anti-CD48 treated mice had a strikingreduction in alveolar space, lung perivascular and peribronchialinflammation and epithelial shedding (FIG. 11A-D). This effect wasspecific to CD48 treatment since anti-CD2 treatment induced a mildinhibitory effect only on the peribronchial inflammatory score, andanti-2B4 treatment seemed to enhance lung inflammation. Importantly,control antibodies did not alter these features.

Example 12 Neutralization of CD48 Attenuates Goblet Cell Hyperplasia,Mucus Production and Smooth Muscle Thickening in the Lung

One of the main features of allergic eosinophilic airway inflammation ismucus production and goblet cell hyperplasia. As assessed by PASstaining, allergen challenge increased goblet cell hyperplasia and mucusproduction. This effect was significantly reduced by anti-CD48pretreatment (FIG. 12A-B) but not anti-2B4 treatment while anti-CD2treatment induced a negligible effect.

In addition, the thickness of the peribronchial smooth muscle layer wassignificantly greater in OVA-challenged mice than in saline-challengedmice. Anti-CD48 mAb-treated mice displayed significantly less smoothmuscle thickening (FIG. 12C). Anti-CD2-treated mice exhibited a minorreduction, while anti-2B4 and control antibodies had no effect.

1. A method of treating an allergic condition, said method comprisingadministering a therapeutically effective amount of an anti-CD48 agent,which blocks CD48 stimulatory pathway, to a subject in need of saidtreatment.
 2. The method according to claim 1, wherein said allergiccondition is an allergic inflammation selected from the group consistingof allergic airway inflammation, atopic dermatitis, conjunctivitis andintestinal allergy.
 3. The method according to claim 2, wherein saidallergic airway inflammation is any one of asthma and nasal polyposis.4. The method according to claim 1, wherein said anti-CD48 agent blocksCD48 stimulatory pathway by inhibiting the activity and/or theexpression of CD48 or of a down-stream member molecule of said pathway,said agent being selected from the group consisting of: a protein basedmolecule which specifically binds to CD48, a nucleic acid-based moleculecomprising at least one target specific sequence complementary to atarget ribonucleic acid sequence comprised within CD48 mRNA and anantagonist molecule of CD48.
 5. The method of claim 4, wherein saidprotein-based anti-CD48 agent is an antibody which specifically binds toCD48 or a CD48 binding fragment of said antibody.
 6. The methodaccording to claim 4, wherein said nucleic acid-based anti-CD48 agent isselected from the group consisting of: an antisense oligonucleotidespecific to the CD48 sequence, a ribonucleic acid molecule selected fromthe group consisting of ribozyme, interfering RNA (RNAi), a smallinterfering RNA (siRNA) and microRNA.
 7. A method of inhibiting theactivity and/or expression of CD48 in cells of a subject in needthereof, said method comprising the step of in vivo contacting saidcells with an effective amount of an anti-CD48 agent selected from thegroup consisting of an anti-CD48 antibody, a CD48 binding fragment of anantibody, an antisense oligonucleotide specific for the CD48 sequence,CD48-specific siRNA, CD48 specific RNAi, CD48-specific Ribozyme, CD48microRNA, any combination thereof and any composition comprising thesame.
 8. A method for the diagnosis of an allergic condition in asubject said method comprising the steps of: (a) obtaining a biologicalsample from said subject; and (b) determining the level of expression ofCD48 in said biological sample by any one of a protein-based or nucleicacid-based detection method; whereby elevated expression of CD48, incomparison with negative control, indicates the presence of saidallergic condition.
 9. The method according to claim 8, wherein saidbiological sample is a body fluid sample selected from the groupconsisting of a leucocyte-containing body fluid, blood, lymph, milk,urine, faeces, semen, appendix, spleen, extractstears, sputum, nasal,mucus, amniotic fluid, bronchoalveolar lavage, pleuric fluid, peritonealfluid and tonsillar tissue extractstears.
 10. The method according toclaim 9, wherein said CD48 expression is detected by a protein-basedmethod comprising the steps of: (i) contacting said sample with a CD48binding agent; and (ii) measuring the level of binding of said agent tosaid CD48 protein; whereby elevated binding of CD48, in comparison witha negative control, indicates the presence of said allergic condition.11. The method according to claim 10, wherein said CD48 binding agent isan anti-CD48 antibody.
 12. The method according to claim 11, whereinmeasuring the levels of binding according to step (ii) is performed by aprotein based detection assay, said assay is any one ofimmunohistochemical staining, Western blot analysis,immunopercipitation, flow cytometry, ELISA, competition assay and anycombination thereof.
 13. The method according to claim 9, wherein saidnucleic-acid based detection method is any one of in-situ hybridization,RT-PCR, nucleic acid based ELISA, RNAse protection assay, Northern blotanalysis and any combinations thereof.
 14. The method according to claim13, wherein said method is an RT-PCR assay comprising the steps of: (a)obtaining a biological sample from said subject; (b) isolating nucleicacids from said sample; (c) providing primers for specific amplificationof CD48 transcripts, together with nucleotides and amplificationreagents; (d) providing conditions for allowing CD48 amplification; (e)performing reversed transcription followed by a PCR amplification onsaid nucleic acids; and (d) determining the level of CD48 amplificationproducts in said sample; whereby elevated levels of CD48 amplificationproducts, in comparison with a negative control, indicates the presenceof said allergic condition.
 15. The method according to claim 8, whereinsaid allergic condition is an allergic inflammation selected from thegroup consisting of allergic airway inflammation, atopic dermatitis,conjunctivitis and intestinal allergy.
 16. The method according to claim15, wherein said allergic airway inflammation is any one of asthma andnasal polyposis.
 17. A kit for the diagnosis of an allergic condition,said kit comprising as follows: (a) an agent for determining thepresence of an analyte of interest, wherein said analyte of interest isone of CD48 protein or CD48 mRNA; and (b) calibration means.
 18. The kitaccording to claim 17, wherein when said analyte of interest is CD48protein, said agent is an anti-CD48 antibody.
 19. The kit according toclaim 17, wherein when said analyte of interest is CD48 mRNA, said agentis one of a CD48-specific primer or a CD48-specific probe.
 20. The kitaccording to claim 17, wherein said allergic condition is an allergicinflammation selected from the group consisting of allergic airwayinflammation, atopic dermatitis, conjunctivitis and intestinal allergy.21. The kit according to claim 20, wherein said allergic airwayinflammation is any one of asthma and nasal polyposis.